Compounds having reduced immunogenicity and a method of reducing the immunogenicity of compounds

ABSTRACT

Reduced-immunogenic fusion compounds are disclosed. The fusion compounds of the invention comprise immunogenic compounds linked to auto-antigenic sequences which render the compound less immunogenic. In addition, a method of reducing the immunogenicity of an immunogenic compound is disclosed. The method comprises linking an auto-antigenic sequence to an otherwise immunogenic compound. Recombinant nucleotide sequence encoding auto-antigenic sequences are also disclosed.

RELATED APPLICATIONS

This application is a continuation of application Ser. No. 08/433,441,filed Jul. 6, 1995, which is the U.S. National Stage of InternationalApplication No. PCT/US93/11148, filed on Nov. 16, 1993, which is aContinuation-in-part of application Ser. No. 07/977,705, filed Nov. 16,1992 (abandoned). The entire teachings of the above applications areincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to fusion compounds having reducedimmunogenicity resulting from the addition to an immunogenic compound ofan amino acid sequence that renders the compound less immunogenic. Theamino acid sequence is found in human proteins. The present inventionrelates to a method of reducing the immunogenicity of compounds by theincorporation of an amino acid sequence, the presence of which resultsin a reduced immune response against the compound.

BACKGROUND OF THE INVENTION

The pharmaceutical use of immunogenic compounds, such as proteins andcarbohydrates, for diagnosis or therapy in humans has enormouspotential. A major concern, however, is that immunogenic compounds oftenelicit immune responses which could limit their effectiveness and, insome cases, cause dangerous allergic reactions. This is particularlytrue of non-human proteins. In addition, it is possible that evenproteins with human amino acid sequences could be immunogenic, as in thecases where the protein is altered in structure or conformation as aconsequence of manufacturing or where the protein is produced in foreignhosts due to inappropriate post-translational modification or improperfolding. Moreover, many non-protein compounds elicit an immune response.

The immune system of the human to whom the immunogenic compound isadministered recognizes the compound as “foreign” and mounts an immuneresponse to remove it. The immune response includes the production ofspecific, high affinity antibodies which bind to and effect eliminationof the immunogenic compound.

Monoclonal antibodies (Mabs) provide examples of the therapeutic uses offoreign proteins. Most Mabs are of murine origin, and have generallybeen found to be immunogenic when injected into humans. Attempts havebeen made to reduce the immunogenicity of murine Mabs by substitutinghuman constant regions for the analogous murine regions to form chimericantibodies or chimeric Mabs, or by going one step further andsubstituting human framework sequences for the murine counterparts inthe variable regions of the antibodies (humanized antibodies orhumanized Mabs). These approaches may reduce the immune responseelicited by murine constant regions or frameworks, but may beineffective in reducing immune responses directed against the variableregions or idiotypes of the Mabs. Indeed, there are several examples ofchimeric Mabs eliciting immune responses directed against the variableregions (for example, B72.3 reported by Meredith, et al., (1992) J.Nucl. Medicine 33:23-29, and ch14.18 reported by Saleh, et al., (1992)Hum. Antibody Hybridoma 3:19-24). In fact, immune responses to theanti-variable region may be the rule rather than the exception. In thesecases, another approach is required.

There is a need for therapeutic or diagnostic compounds which do notelicit either an immune response or which elicit a reduced immuneresponse. There is a need for a method of reducing or eliminating theimmunogenicity of therapeutic and diagnostic compounds.

The present invention provides reduced-immunogenic compounds whichelicit either a reduced immune response or essentially no immuneresponse in humans and a method of reducing the immunogenicity ofcompounds. Reduced-immunogenic compounds according to the presentinvention comprise an auto-antigenic amino acid sequence linked to anotherwise immunogenic protein. By associating an auto-antigenic aminoacid sequence with an immunogenic protein, the human immune systemmounts a reduced immune response against the compound or does not mountan immunogenic response against it all. Accordingly, these compounds canbe administered as therapeutics or diagnostics with a reduction orelimination of the problems associated with the administration ofimmunogenic compounds.

SUMMARY OF THE INVENTION

The present invention relates to reduced-immunogenic fusion compoundswhich comprise immunogenic compounds linked to auto-antigenic sequences.The presence of the auto-antigenic sequence renders the compound lessimmunogenic. In addition, the present invention relates to a method ofreducing the immunogenicity of an immunogenic compound by linking anauto-antigenic sequence to an otherwise immunogenic compound. Thepresent invention also relates to recombinant nucleotide sequence thatencoding auto-antigenic sequences and to essentially pure auto-antigenicpeptides.

DETAILED DESCRIPTION OF THE INVENTION

The presence of antibodies in normal human sera which are specific forportions of degraded proteins, such as portions of endogenous proteinsdegraded by proteolytic enzymes, has been observed. There are manyreports in the literature that refer to observed endogenousimmunoreactivity to cleaved antibody fragments. This endogenousimmunoreactivity to cleaved endogenous proteins is referred to herein as“preimmunity”. The antibodies involved in preimmunity immunoreactivitywere initially described as “agglutinators” or “anti-Fab antibodies”(“αFABA”). It is reported that preimmunity antibodies 1) are present inmost individuals, 2) have varying titers across a population, 3) are notIgM or rheumatoid factors, 4) are fragment specific, and 5) aregenerally of low affinity. These antibodies can be generally describedas a heterogenous group of antibodies that share the characteristic ofrecognizing endogenous protein fragments, usually the terminal portionsof antibody-fragments, which are exposed by protein degradation, usuallyproteolytic degradation.

Osterland, C. K. et al. (1963) Vos Sang 8:133, report a serum activitycapable of “agglutinating” Fab- or F(ab′)₂ coated human erythrocytes.The reactivity is directed to epitopes that only become exposed after animmunoglobulin is cleaved by a proteolytic enzyme. That is, theseantibodies recognize the degraded protein but not the intact protein.

Waller, M. et al., (1969) Immunochemistry 6:207-214, report that“natural antibodies” in human sera were able to differentiate Fabfragments produced by different enzymes. Different antibodies of thisgroup were specific to different epitopes on Fab fragments which weregenerated i.e., exposed, by the specific cleavage that a specific Fabfragment underwent.

Ling, N. R. and P. Drysdale, (1981) Int. Archs. Allergy Appl. Immun.66:459, report that F(ab′)₂ fragments of human, bovine, and rabbitpolyclonal and of human IgG paraproteins of different subclass andlight-chain type were coupled to human red cells and used to detect“agglutinator antibodies” in normal and pathological human sera. Suchantibodies were reported to commonly occur and demonstrate specificityheterogeneity.

Persselin, J. E. and R. H. Stevens, (1985) J. Clin. Invest. 76:723,report that sera from rheumatoid arthritis patients contained twopopulations of antibodies directed against the Fab portion of pooledhuman IgG.

Heimer R., et al., (May 1985) Arthritis and Rheumatism 28(5):562, reportan examination of the specificity of IgG anti-F(ab′)₂ antibodies inunfractionated sera of patients with rheumatoid arthritis and fromaffinity purified antibody preparations.

Persselin, J. E. and R. H. Stevens (1989) Mongr. Allergy 26:74, report agroup of “autologous antibodies” that are directed against the Fab andF(ab′)₂ portions of human IgG. This group, which was reported to beprevalent in normal individuals and patients suffering a variety ofdisorders, was characterized to be a heterogenous group of antibodieswith diverse biological properties and target specificities.

Although many of the reports of “natural antibodies” relate to theexistence of such antibodies that specifically bind to IgG fragments, itis believed that groups these type of antibodies exist which bind todegraded portions of other endogenous proteins.

As used herein, the terms “agglutinators”, “agglutinating antibodies”,“natural antibodies”, “autologous antibodies”, “preimmunity antibodies”and “preimmune serum antibodies” are used interchangeably and are meantto refer to antibodies that are normally present in an individual.Preimmunity antibodies are a heterologous group of antibodies which bindto degraded but usually do not bind to intact endogenous proteins. Theyexist at low levels and generally bind to the terminal portion at acleavage site of a cleaved endogenous protein. There are somepreimmunity antibodies which react to intact proteins. However, manysuch antibodies recognize fragments but do not bind to the intactprotein. In cases in which they do not cross-react with intact proteins,a preimmunity antibody generally recognizes an epitope that occurs atthe terminal portion of a protein following cleavage. This epitopeusually occurs at the C-terminus. By occurring at these positions, theepitope is extremely specific such that the epitope is only accessibleand recognizable when it appears at an end of the protein.

It has been discovered that the presence, on an otherwise immunogeniccompound, of an amino acid sequence which forms the epitope for apreimmunity antibody reduces or eliminates the immunogenicity of thatcompound. The inclusion of such an amino acid sequence allows one toconvert an immunogenic compound into a reduced-immunogenic compound.

As used herein, the terms “auto-antigenic sequence”, “auto-antigenicpeptide”, “preimmunity sequence”, “preimmunity amino acid sequence” and“preimmunity peptide” are used interchangeably and are meant to refer toan amino acid sequence that is an epitope recognized by preimmunityantibody and that, when associated with an immunogenic compound, rendersthe immunogenic compound less immunogenic.

As used herein, the terms “fusion compounds”, “reduced-immunogeniccompound” and “less-immunogenic compounds” are used interchangeably andrefer to compounds which comprise an otherwise immunogenic compoundlinked to an auto-antigenic sequence whereby the presence of theauto-antigenic sequences results in the reduction or elimination of theimmunogenicity of the otherwise immunogenic compound. When animmunogenic compound that is a compound that normally elicits an immuneresponse when administered to a patient, is linked with anauto-antigenic sequence to form a fusion compound, the fusion compoundelicits no immune response or a reduced immune response whenadministered to a patient compared to the immune response elicited bythe immunogenic compound by itself.

As used herein, the term “less immunogenic” refers to the comparativelyreduced-immunogenicity exhibited by an immunogenic compounds linked toan auto-antigenic sequence relative to the immunogenicity exhibited bythe same immunogenic compound which is not linked to an auto-antigeniccompound.

As used herein, the term “immunogenic” refers to the ability of acompound to elicit an immune response.

As used herein, the term “antigenic” refers to the ability of a compoundto react with the immune system, i.e. antibodies.

Auto-antigenic sequences can be identified by a variety methods whichcan be readily performed by those having ordinary skill in the art.Endogenous proteins, such as antibodies, cytokines, growth factors,receptors, enzymes and structural proteins, can be cleaved by a panel ofproteolytic enzymes. The fragments produced can be exposed to human seraand those fragments that bind to preimmunity antibodies in the sera canbe readily identified. The amino acid sequence of the epitope that isinvolved in the preimmunity antibody can be determined. This epitoperepresents an auto-antigenic sequence. Alternatively, peptide librariesthat contain a random assortment of peptides of about 5 or more aminoacid residues can be produced. These libraries can be used in a screenwith normal human sera to identify peptides that are epitopes recognizedby endogenous preimmunity antibodies. These peptides can be identifiedand used tested as auto-antigenic sequences.

One example of a heterogeneous group of preimmunity antibodies specificfor protein degradation products are antibodies which recognize epitopesthat occur on the heavy chain C-terminal sequence when IgG antibodiesare degraded by proteolytic cleavage. Accordingly, some embodiments ofthe present invention relate to a method of rendering an otherwiseimmunogenic compound less-immunogenic by linking to it one of a varietyof auto-antigenic sequences found at the heavy chain C-terminus of humanor chimeric Fab or F(ab′)₂ molecules.

Amino acid sequences that are the epitopes for anti-IgG fragmentpreimmunity antibodies are often found at the hinge region of the heavychain. Cleavage of the heavy chain at the hinge region generates anamino acid sequence at the C-terminus which may be recognized byspecific preimmunity antibodies that do not react with intact IgGmolecules. Depending upon where cleavage occurs, a different epitope isexposed and thus a different set of preimmunity antibodies may bind toit. Thus, individual antibodies of this group recognize the discreetepitopes produced by cleavage at various sites.

In some embodiments, the auto-antigenic sequence is derived from thehinge region of IgG. In some preferred embodiments, the auto-antigenicsequences is derived from the hinge region of IgG₁.

The hinge region refers to various structural segments of the heavychain of IgG molecules. Different classes of IgG molecules havedifferent amino acid sequences at their respective hinge regions. Thehinge region is a particularly variable element of immunoglobulinstructure. Table 1 provides a listing of the different amino acidsequences of the respective hinge regions of the various classes of IgGmolecules.

In order to identify whether a particular amino acid sequence is anauto-antigenic sequence, IgG molecules can, for example, be cleaved withone of a panel of proteases to provide IgG fragments that containdifferent hinge region sequences at the terminal end. Alternatively,peptides and polypeptides can be produced by peptide synthesis orrecombinant DNA technology which are modelled upon the sequence of thehinge region. In either case, human sera can be screened to determinewhether preimmunity antibodies are present which bind to a particularexposed terminal sequence or synthetic peptide, respectively.

It has been observed that the amino acid sequence near the papaincleavage site of human Fab molecules is reactive with the endogenoushuman “anti-Fab” preimmunity antibodies. This sequence can instruct theimmune system to ignore a molecule that includes it such that no furtherimmune response is elicited. This auto-antigenic sequence prevents animmune response to a linked compound that would be otherwiseimmunogenic.

A Fab-derived preimmunity sequence having the C-terminal sequence CDKTH(SEQ ID NO:1) was identified from observations made concerning thenature of preexisting human immunity and induced immune responses tomurine and chimeric 7E3 Fab fragments. Both the light and heavy chainsof the chimeric 7E3 Fab comprises of murine variable regions and humanconstant regions. This sequence mimics a natural fragment orconformation of human IgG found in human serum, and therefore, a typicalhigh affinity immune response is not mounted against the molecule, oragainst the related Fab sequence. A reduced immunogenicity of the murine7E3 variable region when linked to this sequence in the c7E3 moleculewas observed. According to the invention, an immunogenic compound, suchas a foreign protein, may be rendered non-immunogenic or lessimmunogenic by linking an auto-antigenic sequence, such as those foundat the C-terminus of papain generated human Fab molecules derived fromIgG₁, to a foreign compound. Thus, association of an auto-antigenicsequence with an immunogenic therapeutic or diagnostic agent is usefulfor reducing the immunogenicity of the therapeutic or diagnostic agent,thereby preventing or reducing a significant immune response to theagent when administered to a patient. Similarly, papain generated Fabfragments of chimeric antibody c128 which is specific for CD4 and papaingenerated Fab fragments of chimeric antibody c168 which is specific fortumor necrosis factor have the preimmunity sequence, CDKTH (SEQ IDNO:1), at their the C-termini.

In addition, cleavage of antibodies with human constant regions such asc128, c168 or c7E3 with elastase exposes a preimmunity sequence at the Ctermini of the antibody fragment thus generated.

The F(ab′)₂ fragment of chimeric 7E3 has also been found to exhibit asimilar characteristic to the Fab molecule, such as showing a naturalimmunity, or preimmunity, in normal human sera. Therefore, theauto-antigenic sequence at its C-terminal sequence may also be usefulfor reducing the immunogenicity of foreign compounds in humans.

Preferred auto-antigenic sequences may comprise amino acid sequencesselected from the group consisting of: CDKTH (SEQ ID NO:1), PKSCD (SEQID NO:2), KSCDK (SEQ ID NO:3), SCDKT (SEQ ID NO:4), DKTHT (SEQ ID NO:5),KTHTC (SEQ ID NO:6), THTCP (SEQ ID NO:7), HTCPP (SEQ ID NO:8), TCPPC(SEQ ID NO:9) and CPPCP (SEQ ID NO:10). These sequences can beassociated with the otherwise immunogenic compound in such a way as toensure that the last residue of each particular sequence represents aC-terminal amino acid residue.

In order to determine whether an amino acid sequence will be useful asan auto-antigenic sequence, peptide constructs comprising the specificsequence to be tested are exposed to human sera to determine whetherantibodies are present in the sera which recognize and specifically bindto the sequence.

The most preferred auto-antigenic sequence comprises the amino acidsequence CDKTH (SEQ ID NO:1). As noted above, the H residue is theC-terminal residue of any construction which contains this peptide. Whenassociated with a compound and present at the C-terminal region, thispeptide reduces or eliminates the immunogenicity of the compound, thusrendering the compound less-immunogenic.

Once identified, an auto-antigenic sequence can be linked to animmunogenic compound by a variety of means that can be readily practicedby those having ordinary skill in the art. If the immunogenic compoundis a protein, a fusion protein comprising the auto-antigenic sequence atthe terminal portion of the immunogenic protein can be produced usingrecombinant DNA technology. A nucleotide sequence that encodes anauto-antigenic sequence can be linked to the nucleotide sequence thatencodes the immunogenic protein to form a chimeric gene that encodes afusion protein. The auto-antigenic sequence will appear at the terminalportion of the resulting fusion protein when the chimeric gene isexpressed. If the immunogenic compound is not a protein, syntheticpeptides that comprise the auto-antigenic sequence at a terminus can beproduced by standard methodology. These peptides can be chemicallylinked to the immunogenic compound using well known techniques.Auto-antigenic sequences can also be linked to proteins by chemicalmeans. Regardless of the method of linking an auto-antigenic sequence toan otherwise immunogenic compound, the immunogenic compound is convertedto a reduced-immunogenic compound by the incorporation of anauto-antigenic sequence which serves as an epitope for preimmunityantibodies. One having ordinary skill in the art can accomplish linkageof an auto-antigenic sequence to an immunogenic compound by well knowntechniques. Standard coupling techniques, for example, include but arenot limited to: coupling through free sulfhydryl of cysteine, couplingthrough ε-amino group of lysine and coupling through any free amine.Techniques for engineering antibodies are well known and described inWinter and Millstein (1991) Nature 349:293, and Larrich and Fry (1991)Hum. Antibod. and Hybridomas 2:17, both of which are incorporated hereinby reference.

According to the invention, an auto-antigenic sequence can be attachedto an immunogenic compound in order to convert the immunogenic to areduced-immunogenic compound. As used herein, the terms“reduced-immunogenic compounds”; “less-immunogenic compounds”,“non-immunogenic compounds” and “fusion compounds” are usedinterchangeably and meant to refer to compounds which comprise anauto-antigenic sequence linked to an otherwise immunogenic compound. Thepresence of the auto-antigenic sequence linked to the otherwiseimmunogenic compound causes a reduced immune response in individualsadministered such compounds relative to the immune response elicited bythe immunogenic compound absent the auto-antigenic sequence. Anauto-antigenic sequence may be added to non-human proteins, processed orrecombinantly produced human proteins or non-protein immunogeniccompounds.

Examples of non-human proteins include, but are not limited to, Mabs,Fabs, F(ab′)₂s, non-human cytokines, non-human growth factors, non-humanreceptors, non-human structural proteins and non-human enzymes such asStreptokinase.

Examples of processed or recombinantly produced human proteins include,but are not limited to, human and chimeric antibodies and fragmentsthereof, human cytokines, human growth factors, human receptors, humanstructural proteins and human enzymes such as coagulation andfibrinolytic agents.

Examples of non-protein immunogenic compounds include, but are notlimited to, carbohydrates such as heparin.

A preferred immunogenic compound to be converted to areduced-immunogenic compound according to the present invention is amurine IgG molecule. Ordinarily, a murine IgG will elicit an immuneresponse when administered to a human. This response can render itineffective or less effective because the IgG molecule is neutralizedand/or removed prior to reaching and binding to its target antigen. Byrendering the murine IgG less-immunogenic, it becomes more effective asa therapeutic or diagnostic since it is less deterred by the patient'simmune system.

A preferred embodiment of the present invention is a murine IgG moleculehaving an auto-antigenic sequence comprising CDKTH (SEQ ID NO:1) linkedsuch that the H residue is a C-terminal residue. According to theinvention, such a molecule can be produced by standard recombinant DNAtechniques used to produce antibodies. For example, a nucleotidesequence encoding the auto-antigenic sequence can be inserted at the 3′end of a gene encoding a C-terminal portion of the IgG molecule,preferably the C-terminal portion of the heavy chain. The nucleotidesequence is inserted in the proper reading frame such that the residuesencoded by it will occur at the very end of the resulting protein.

Clinical results demonstrate a reduction the immunogenicity to foreignantigens containing an auto-antigenic sequence that is exposed byproteolytic cleavage of an IgG heavy chain. Several observations havebeen made during the development of the therapeutic anti-platelet Mab7E3 which led to the discovery that the immunogenicity of a normallyimmunogenic compound may be reduced by associating it with an amino acidsequence which represents an epitope recognized by a preimmunityantibody. These experiments are reported in Example 1.

Briefly, Mab 7E3 was injected into humans both as a murine Fab fragmentand as a chimeric Fab fragment (c7E3). The immunogenicity of bothfragments were analyzed. There was a fundamental difference in thenature of the immune responses elicited by the murine and chimeric Fabs.The murine 7E3 Fab elicited an immune response in patients which wasdirected almost entirely against the 7E3 variable region. In contrast,even though the c7E3 Fab contained the identical variable region asmurine 7E3 Fab, c7E3 did not elicit comparable immune responses,indicating that the human constant region of the c7E3 Fab rendered thevariable region less immunogenic.

Direct-coated, affinity-independent EIA analyses indicated that 50-80%of the normal human population has preimmune serum antibodies that reactwith chimeric Fab fragments. This preimmunity reactivity is not specificfor the variable regions of these molecules since various monoclonalchimeric Fab fragments as well as bulk Fab fragments prepared from totalhuman serum Ig were recognized by the endogenous anti-Fab antibodies.This anti-fragment reactivity appeared to be of low affinity, and wasdetectable only by relatively sensitive, solid phase EIA assays.

The location of the reactive epitopes of the monoclonal chimeric (orhuman) Fabs was at the C-terminus of the heavy chain of the Fab fragmentgenerated by papain digestion of the intact IgG molecule. Theseendogenous anti-chimeric 7E3 Fab preimmunity antibodies were readilyneutralized using the Fab fragment of another IgG₁ chimeric antibody butnot by other proteins. These observations indicated that the reactiveepitope is probably a short sequence of amino acids which mustbe-present at or near the C-terminus of the Fab fragment.

A human or chimeric Fab fragment mimics a molecule which is normallyfound in serum and which elicits a normal low affinity antibodyresponse. It appears that the immune response against molecules thatcontain the auto-antigenic sequence as an accessible epitope may beregulated to preclude a high affinity secondary response. In effect, theimmune system may be desensitized or tolerized to antigen challenge withmolecules bearing the epitope. Further challenge, therefore, with asimilar molecule would not lead to a typical high affinity immuneresponse.

Preimmunity to particular epitopes appears to be species-specific. Whenthe immune responses from primates treated with chimeric 7E3 Fab wereanalyzed, pretreatment sera from these monkeys demonstrated little or noimmunoreactivity to chimeric 7E3 Fab but showed a significant reactivityto Fab fragments generated from monkey IgG. Conversely, human serashowed no preexisting immunoreactivity to monkey Fab. In addition,monkeys have demonstrated a significantly greater induced immunogenicityto chimeric 7E3 Fab than have the humans enrolled Phase I clinicaltrials.

Other species were also examined for preexisting immunoreactivity totheir autologous Fab and F(ab′)₂ fragments. Autologous panels of serafrom rabbits and goats were screened for reactivity to polyclonal Faband F(ab′)₂ fragments from IgG of their respective species. Again, itwas observed that there was significant IgG reactivity to both of theseantibody fragments from their own species. The same observation,however, was not made for murine antibodies to murine 7E3 Fab.

Although comparative experiments on humans have not been performed usingmurine and chimeric 7E3 F(ab′)₂ fragments or other murine and chimericFab and F(ab′)₂ fragments, specific antibodies which bind to human Faband F(ab′)₂ fragments have been observed. The individuals with highanti-chimeric Fab reactivity do not necessarily have high anti-1-5chimeric F(ab′)₂ reactivity, and vice versa. Furthermore, the immunerecognition of these chimeric fragments is apparently specific, sincethe binding of anti-Fab antibodies generally cannot be blocked bychimeric Fab′ or F(ab′)₂.

The description of the present invention is generally presented hereinas relating to compounds which are non-immunogenic as a result oflinking a human auto-antigenic sequence to an otherwise immunogeniccompound, and to a method of reducing the immunogenicity of compounds bylinking the immunogenic compound with a human auto-antigenic sequence.It is contemplated that the present invention can be applied to otherspecies. The scope of the present invention is intended to include tocompounds which are less-immunogenic in a particular species as a resultof linking an auto-antigenic sequence form the species to an otherwiseimmunogenic compound and to a method of reducing the immunogenicity ofcompounds in a particular species by linking the immunogenic compoundwith an auto-antigenic sequence from that species.

Furthermore, the description of the present invention is presented asrelating to compounds in which an auto-antigenic sequence is physicallyattached to an otherwise immunogenic compound. However, it is possiblethat physical linkage is not required. It is contemplated that reducedimmunogenicity of a protein can be achieved by co-injection of an autoantigenic peptide sequence or a non-specific human Fab fragments.

EXAMPLES Example 1

The 7E3 monoclonal antibody has been developed for human therapy as aFab fragment of the antibody molecule. The reagent was produced in twoversions; one derived from the murine monoclonal antibody (murine gamma1 isotype) by papain digestion (m7E3), and one derived from amouse/human chimeric monoclonal antibody (human gamma 1 isotype) also bypapain digestion (c7E3). The chimeric Mab was produced using standardcloning techniques to obtain the variable region genes from the 7E3hybridoma and fuse them to previously cloned human constant region genesin vitro. The chimeric genes were introduced into appropriate mammaliancells for expression.

The two Fab molecules have the same variable region, but differentconstant regions. The C-terminal amino acid sequences exposed afterpapain digestion are different. Papain clips the antibody molecules inthe hinge region of the heavy chain between the CH1 and CH2 domain. Thehinge amino acid sequences of the human gamma 1 and the mouse gamma 1are very different (See Table 1). After cleavage of c7E3 with papain,the C-terminus of the heavy chain ends with the amino acids CDKTH (SEQID NO:1). The exact papain cleavage point in the mouse gamma 1 hinge isunknown, but must produce a very different C-terminus from the humansequence, since the mouse hinge does not contain a similar sequence toCDKTH (SEQ ID NO:1).

The m7E3 and c7E3 Fab fragments were tested to determine whether humansera contains antibodies which react with the fragments. A solid phaseELISA assay was used in which either m7E3 Fab or c7E3 Fab wasimmobilized directly on plastic assay plate and exposed to human serum.Bound human antibodies were detected using goat anti-human antibodiesconjugated to an enzyme that will produce a colored product whenincubated with the appropriate substrate. This assay is very sensitiveand capable of detecting low affinity interactions. A large number ofhuman sera were tested, and the m7E3 Fab was generally non-reactive,whereas c7E3 Fab reacted with at least 60% of the human serum samples.

The specificity of the reactive antibodies in human serum was assessedby including an excess of various molecules in the assay as competitiveinhibitors. If a given molecule inhibits binding to c7E3, then it mustalso display the reactive epitope(s). As expected, soluble c7E3 couldinhibit the binding of the human antibodies to immobilized c7E3. Othermolecules that could inhibit the binding included Fab fragments of otherchimeric gamma 1 antibodies, and polyclonal human Fab derived fromserum. In contrast, none of the following molecules could inhibit thebinding: 1) m7E3 Fab; 2) whole c7E3 IgG; 3) Fab fragment derived fromIgG₄ version of 7E3 (contains different hinge region from gamma 1; 4)F(ab′)₂ fragment of c7E3; and 5) Fab′ fragment of c7E3 Fab fragment isextremely specific for the C-terminus exposed after papain digestion ofthe c7E3 antibody.

The immunogenicities of m7E3 Fab and c7E3 Fab were tested in humans inPhase I clinical trials. Using an assay format that minimizes thelow-affinity preimmune reactivity so that actual treatment-relatedresponses can be easily seen, it was found that 17/86 or about 20% ofthe subjects receiving m7E3 Fab exhibited immune responses at titersranging from 1:50-1:1600. In contrast, only 1/67 or 1.5% of the subjectsreceiving c7E3 Fab exhibited an immune response (titer=1:50). The c7E3Fab, which reacts with endogenous human antibodies, was therefore shownto be must less immunogenic than m7E3 Fab, which does not react withendogenous antibodies.

Example 2

The possible sequences derived from the hinge region of human gamma 1Fab (and F(ab′)₂) reactive with endogenous “anti-fragment” preimmunityantibodies can be identified by screening and competition experimentsusing synthetic peptides. Synthetic peptides of about 5 amino acids ormore in length are produced which contain various sequences found in theamino acid sequence of the hinge region.

The most reactive sequences may then be linked to an immunogeniccompound of interest to reduce immunogenicity. Linkage can beaccomplished in several ways. If the immunogenic compound is a protein,the natural C-terminus of the immunogenic protein can be converted tothe desired sequence by site-directed mutagenesis of the gene, mosteasily accomplished by using appropriately designed PCR primers todelete the natural termination codon and add the desired sequence. Thesetechniques could be used to add or substitute the sequence at anyposition in any gene. Alternatively, a synthetic peptide can beconstructed corresponding to the minimal reactive-sequence and couldlinked to the immunogenic compound by chemical means.

Example 3

Amino acid sequences that are reactive with antibodies in normal humanserum, which may be used as auto-antigenic sequences to render foreignor normally immunogenic molecules less-immunogenic, may be identified byscreening synthetic peptides with human sera. This is a general methodwhich does not require identification of a protease-specific cleavagesite of an endogenous protein, and does not even require that the actualsequence occur naturally.

Briefly, a library of random peptides of at least about 5 or more, andpreferably about 5-10 amino acids in length is generated. These peptidesare screened for reactivity with antibodies in human sera. This isaccomplished, for example, by immobilizing the peptides on a solid phaseand performing a standard ELISA assay to detect bound human antibodiesafter exposure to sera.

Positive peptides are then evaluated further to establish if they arereactive with human sera only and not with other species, and todetermine the affinity of the interactions. Peptides that react with themajority of human sera but not sera of other species, are then studiedto determine whether they are capable of conferring reducedimmunogenicity on otherwise immunogenic compounds. Whether or not agiven peptide elicits an immune response requires empiricalimmunogenicity data.

There are many possible ways to generate and screen libraries or randompeptide sequences. One method is to synthesize collections of peptides(Schoofs, P. G. et al. (1988) Immun. 140:611) and assay pools forreactivity against human serum. Positive pools are subdivided andreassayed multiple times so that the active species would eventually beidentified. Another method is to generate DNA sequence that codes forrandom amino acid segments and fuse the DNA sequences to a bacteriophagegene (McCafferty et al., (1990) Nature 348:552). The random amino acidsequences are-displayed on the outside of the bacteriophage particle asan artificial C-terminus of a phage protein. The phage is immobilizedand screened for reactivity to endogenous human antibodies. Positivephage are isolated and the DNA extracted and sequenced to determine theamino acid sequence of the reactive peptide segment.

Example 4

In general, it is practically difficult to test auto-antigenic sequencecandidates in humans to determine whether or not their presence rendersimmunogenic compounds less immunogenic because it requires experimentaltesting in humans which raises ethical concerns in many circumstances.Because of this difficulty in testing the immunogenicity of candidatemolecules in humans, a rabbit model is used to mimic the human systemand demonstrate that epitopes of preimmunity antibodies can beidentified and linked to immunogenic compounds to reduce theimmunogenicity of the compounds.

Epitopes of preimmunity antibodies can be identified as described inExample 3 but substituting rabbit sera for the human sera used in theprotocols described therein. Amino acid sequences that react with theendogenous antibodies can be identified and linked to various compoundsby well known methods. Comparative data can be generated fromexperiments in which the compounds linked to the suspectedauto-antigenic sequences and compounds alone are administered torabbits. The immune response against each of the compounds can bemeasured.

Rabbits have been shown to exhibit the same type of normal reactivity tohomologous Fab fragments; that is, preimmunity to Fab fragments. Theappropriate region of rabbit DNA from the immunoglobulin heavy chainlocus is cloned and the sequence of the hinge region is determined. Thisallows the construction of fusion compound which comprise a rabbitauto-antigenic sequence linked to the chimeric 7E3 IgG at the C-terminusof a Fab molecule and evaluating its immunogenicity compared to theimmunogenicity of chimeric 7E3 Fab in rabbits.

Example 5

Streptokinase is a thrombolytic protein which has been approved as adrug for heart attack patients suffering coronary blockages. One majordisadvantage of Streptokinase is that it is highly immunogenic inhumans. Accordingly, its usefulness is greatly limited. Particularly,once a patient has been administered Streptokinase, there is the chanceof dangerous immune reaction to any subsequent administration.

The present invention provides a method of reducing the immunogenicityof Streptokinase, thereby reducing the inherent shortcomings of themolecule as a therapeutic which forms the basis for safety concerns thatare associated with its use.

Streptokinase production using recombinant DNA technology is well knownand can be performed by those having ordinary skill in the art usingreadily available starting materials. Similarly, well known techniquesmay be performed to produce a Streptokinase derivative that has theauto-antigenic sequence CDKTH (SEQ ID NO:1) at the C-terminus of theprotein. One having ordinary skill in the art can produce such aStreptokinase derivative without undue experimentation.

Streptokinase derivative can be tested in vitro and in animal models toensure that it retains Streptokinase activity. Comparative clinicalexperiments can then be performed to measure the immunogenicity ofStreptokinase versus the immunogenicity of Streptokinase derivative. Thetechniques to perform these experiments are well known and can bereadily performed by those having ordinary skill in the art.

Example 6

There are several examples of chimeric Mabs which elicit immuneresponses directed against the variable regions. These include B72.3(Meredith et al., (1992) J. Nucl. Medicine 33:23-29) and ch14.18 (Saleh,et al., (1992) Hum. Antibody Hybridoma 3:19-24). The fact that theseantibodies elicit immune responses represents a major obstacle in theireffectiveness and their usefulness is greatly limited by theirimmunogenicity. Particularly, patients develop antibodies against theantibodies and neutralize their activity.

The present invention provides a method of reducing the immunogenicityof chimeric Mabs, thereby reducing the basis for safety concerns thatare associated with its use and increasing their utility. Techniques forengineering antibodies are described in Winter and Millstein (1991)Nature 349:293, and Larrich and Fry (1991) Hum. Antibod. and Hybridomas2:17. Production of B72.3 and ch14.18 can be accomplished from readilyavailable starting materials using recombinant DNA technology well knownby those having ordinary skill in the art. Likewise, well knowntechniques may be performed to produce chimeric Mab derivatives dB72.3and dch14.18 in which the auto-antigenic sequence CDKTH (SEQ ID NO:1) ispresent at the C-terminus of each antibody respectively, preferably theheavy chain. One having ordinary skill in the art can produce such achimeric Mabs and chimeric Mab derivatives without undueexperimentation.

Chimeric Mab derivatives dB72.3 and dch14.18 can be tested in vitro andin animal models to ensure that they retain their specificity andactivity. Comparative clinical experiments can then be performed tomeasure the immunogenicity of chimeric Mabs B72.3 and ch14.18 versus theimmunogenicity of chimeric Mab derivatives dB72.3 and dch14.18,respectively. The techniques to perform these experiments are well knownand can be readily performed by those having ordinary skill in the art.TABLE 1 Hinge Sequences of Human and Mouse Immunoglobulins Antibody typeHinge Sequence Human IgG₁ EPKSCDKTHTCPPCP (SEQ ID NO:11) Human IgG₂ERKCCVECPPCP (SEQ ID NO:12) Human IgG₃ ELKTPLGDTTHTCPRCP (SEQ ID NO:13)Human IgG₃M15 EPKSCDTPPPCPRCP (SEQ ID NO:14) Human IgG₄ ESKYGPPVPRDCG(SEQ ID NO:15) Mouse IgG₁ EPRGPTIKPGPSCP (SEQ ID NO:16) Mouse IgG_(2a)CKPCICTCPPCKCP (SEQ ID NO:17)

1. A fusion compound comprising an immunogenic compound linked to theamino terminal of an auto-antigenic sequence, the carboxy terminal ofthe auto-antigenic sequence being unlinked, with the proviso that thefusion compound is not c7E3 Fab.
 2. The fusion compound of claim 1wherein said auto-antigenic sequence is an IgG amino acid sequence. 3.The fusion compound of claim 1 wherein said auto-antigenic sequence isan IgG heavy chain amino acid sequence.
 4. The fusion compound of claim1 wherein said auto-antigenic sequence comprises amino acid sequencesselected from the group consisting of: SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO: 4 r SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ IDNO:8, SEQ ID NO:9 and SEQ ID NO:10.
 5. The fusion compound of claim 1wherein said auto-antigenic sequence comprises SEQ ID NO:1.
 6. Thefusion compound of claim 1 wherein said immunogenic compound is aprotein.
 7. The fusion compound of claim 1 wherein said immunogeniccompound is a non-human protein.
 8. The fusion compound of claim 1wherein said immunogenic compound is a murine monoclonal antibody. 9.The fusion compound of claim 1 wherein said immunogenic compound is achimeric monoclonal antibody.
 10. The fusion compound of claim 1 whereinsaid immunogenic compound is Streptokinase.
 11. The fusion compound ofclaim 1 wherein said immunogenic compound is a monoclonal antibody andsaid auto-antigenic sequence is SEQ ID NO:1.
 12. A method of reducingthe immunogenicity of an immunogenic compound comprising the step oflinking said compound to the amino terminal of an auto-antigenicsequence, the carboxy terminal of the auto-antigenic sequence beingunlinked, with the proviso that the fusion compound is not c7E3 Fab. 13.The method of claim 12 wherein said auto-antigenic sequence is an IgGamino acid sequence.
 14. The method of claim 12 wherein saidauto-antigenic sequence is an IgG heavy chain amino acid sequence. 15.The method of claim 12 wherein said auto-antigenic sequence comprisesamino acid sequences selected from the group consisting of: SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10.
 16. The method of claim12 wherein said auto-antigenic sequence comprises SEQ ID NO:1.
 17. Themethod of claim 12 wherein said immunogenic compound is a protein. 18.The method of claim 12 wherein said immunogenic compound is a non-humanprotein.
 19. The method of claim 12 wherein said immunogenic compound isa murine monoclonal antibody.
 20. The method of claim 12 wherein saidimmunogenic compound is a chimeric monoclonal antibody.
 21. The methodof claim 12 wherein said immunogenic compound is Streptokinase.
 22. Themethod of claim 12 wherein said immunogenic compound is a monoclonalantibody and said auto-antigenic sequence is SEQ ID NO:1.
 23. The methodof claim 12 wherein said auto-antigenic sequence is linked to saidimmunogenic compound by chemically binding a peptide comprisingauto-antigenic sequence to said immunogenic compound.
 24. The method ofclaim 12 wherein said immunogenic compound is a protein and saidauto-antigenic sequence is linked to said immunogenic protein by bindinga nucleotide sequence that encodes said auto-antigenic sequence to the3′ terminal end of nucleotide sequence that encodes said immunogenicprotein to form a chimeric gene, expression of said chimeric geneproduces a fusion protein having said auto-antigenic sequence linked tosaid immunogenic protein.
 25. A recombinant nucleic acid moleculeconsisting a nucleotide sequence encoding an auto-antigenic sequence.26. The recombinant nucleotide sequence of claim 25 wherein saidauto-antigenic sequence comprises SEQ ID NO:1.
 27. The recombinantnucleotide sequence of claim 25 further comprising a nucleotide sequenceencoding an immunogenic protein wherein the 5′ end of said nucleotidesequence that encodes an auto-antigenic sequence is linked to the 3′ endof said nucleotide sequence that encodes said immunogenic protein, the3′ end of said nucleotide sequence that encodes the auto-antigenicsequence being linked to a termination sequence without any interveningcoding sequences, said immunogenic protein is a monoclonal antibody andsaid auto-antigenic sequence is SEQ ID NO:1.